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The biochemical anatomy of cortical inhibitory synapses.

Abstract
Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.

Figure 4. Biochemical purification of a tagged inhibitory synaptic protein complex.(A) Immunoblotting of various proteins shows that detergent solubilized protein extract S3 was enriched in both inhibitory (VGABAARα1, GABAARα1, GABAARβ2/3, GABAARγ2) and excitatory (GluR2, PSD95) synaptic proteins, as well as mitochondria (COx). Gel filtration of fraction S3 enabled enrichment of synaptic protein complexes relative to intracellular proteins, as shown by the specific exclusion of the endoplasmic reticulum marker BIP, from the high molecular weight fractions (6–10). Protein concentration of each fraction was measured (top), and the void volume determined by the elution of Blue Dextran (2000 kDa). Identical results were obtained for endogenous proteins in fractions prepared from wildtype or Otx1-eGFP cortices (not shown). (B) Fractions 6–10 (red box in A) from Otx1-VGABAARα1 or Otx1-eGFP control were pooled and subject to co-immunopurification using an anti-eGFP antibody. Immunoblotting confirmed the specific presence of inhibitory synaptic proteins (VGABAARα1, GABAARα1, GABAARβ2/3, GABAARγ2) and the absence of excitatory synaptic (GluR2, PSD95) and mitochondrial (COx) proteins in the material immunopurified via VGABAARα1. Only soluble eGFP was detected in the control sample. IN: Input. FT: Flow-through. IP: Immunoprecipitate. V: Venus. GAR: GABAA receptor. Further biochemical experimental results are presented in Figure S1.

Figure 5. Mass spectrometry identifies proteins present at tagged inhibitory synapses.(A) All peptides were evaluated individually, for their presence or absence in the sample isolated via VGABAARα1 or eGFP, using information from peptide fragmentation spectrum (MS/MS), peptide mass spectrum (MS), and peptide retention time in extracted ion chromatogram. An example is shown for peptide, GDDNAVTGTK, from GABAARβ2. V: Venus. GAR: GABAA receptor. (B) Schematic representation of the cortical inhibitory synaptic protein complex. These synapses contain a multitude of inhibitory receptors, as well as cell signaling and adhesion proteins, but are entirely lacking in cell signaling molecules. The localization of LHFPL4 and Neurobeachin is hypothetical. Complete information on each peptide is in Table 1 and Figure S2 and Table S1.

Figure 6. Proteins identified by mass spectrometry are present at inhibitory synapses.(A) Immunoblotting of several proteins identified by mass spectrometry confirmed their presence in immunopurified inhibitory synapses. GABAARα2, neuroligin2 and neuroligin3 are present, while excitatory markers neuroligin1 and homer are absent from VGABAARα1-tagged inhibitory synapses. The abundant signaling molecule CaMKII is also absent. (B-F) Immunofluorescence studies confirm the colocalization of several proteins identified by mass spectrometry with VGABAARα1. Gephyrin is localized to inhibitory synapses on both the cell soma and axon initial segment (B), while PSD95 is markedly absent (C). GABAARβ2/3 (D), Nlgn2 (E) and Nlgn3 (F) also colocalize with VGABAARα1 in cortical pyramidal neurons. Scale: 10 µm. V: Venus. GAR: GABAA receptor.